The present invention relates to cloning nucleic acid molecules that have been exposed to an enzyme, or other substance, which produces an overhanging nucleotide(s) at one or more 3xe2x80x2 or 5xe2x80x2 ends of the molecules.
The polymerase chain reaction (PCR) has become an invaluable tool for molecular biologists. Numerous applications have been discovered for PCR. Examples include DNA fingerprinting, DNA sequencing, site directed mutagenesis and cloning applications.
PCR is based on three discrete, multiply repeated steps: denaturation of a DNA template, annealing of a primer to the denatured DNA, and extension of the primer with a polymerase to create a nucleic acid complementary to the template. Each primer extension product specifically anneals with a complementary primer and the resulting primed template DNA acts as a substrate for a further extension reaction. These steps are repeated many times, for example, by using an automated cycling procedure, thereby exponentially amplifying the initial nucleic acid material. The conditions under which these steps are performed are well established in the art. Procedures for conducting PCR have been extensively described. See, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202, which are incorporated in their entirety herein by reference.
It is often desirable to obtain clones of a PCR amplified DNA product. A common method for cloning PCR products involves incorporation of flanking restriction sites onto the ends of primer molecules. In this method the PCR cycling is carried out and the amplified DNA is purified, restricted with an appropriate endonuclease(s) and ligated to a compatible vector preparation. Thus, this PCR cloning method requires the step of preparing PCR primer molecules which include base sequences having a preferred restriction recognition sequence. Also, this method can result in unintended internal restriction of uncharacterized restriction site sequences. In addition, cleavage of the PCR product by the preferred restriction enzyme may often be inefficient because the restriction site is close to the end of the DNA molecule to be cleaved. Such limitations add to the cost and complexity of cloning PCR products on a routine basis.
Many DNA polymerases, including Taq DNA polymerase and other thermostable DNA polymerases may catalyze a non-template directed addition of single deoxynucleotide monophosphate (dNMP) residue to the 3xe2x80x2 termini of blunt-ended DNA duplexes. Thus, DNA polymerase may naturally create a single base, cohesive termini on otherwise blunt end DNA fragments. For example, during PCR, a thermostable DNA polymerase used may add a single nucleotide to the 3xe2x80x2 end of an otherwise blunt end DNA PCR product. These overhanging residues are widely viewed as incompatible with most molecular cloning schemes. The overhanging residues may be removed with enzymes such as S1 nuclease and Klenow fragment to create blunt ends. The blunted PCR products can then be cloned into a vector which has been digested with a restriction enzyme that will leave a blunt end compatible with the blunted PCR fragment.
Many problems exist with blunt end cloning of PCR products. For example, enzymatic blunting of the PCR product may often be inefficient. Also, recircularization of the blunt ended linear vector with no target sequence inserted is common leading to a low cloning efficiency. To help overcome this last problem dephosphorylation is sometimes used.
Dephosphorylation of the vector can help prevent self-ligation thereby increasing the efficiency of target sequence insertion. However, this procedure can be problematic because of, for example, the difficulty that may be involved in inactivating the phosphorylase.
One technique that has been developed to directly clone PCR products takes advantage of the Taq polymerase catalyzed non-template directed addition of dAMP residue to the 3xe2x80x2 termini of some blunt-ended DNA PCR products (described in U.S. Pat. Nos. 5,487,993 and 5,827,657). This technique uses a linearized vector with compatible, cohesive, single nucleotide dTMP residues overhanging each end of the linearized vector. PCR DNA products which have a dAMP nucleotide overhang at both 3xe2x80x2 ends may be cloned into these vectors. This method is based on the publication of Clark (Nucleic Acids Res. Vol. 16 pg. 9677-9686). In Clark, the data showed that a dAMP nucleotide was preferentially added to an 18 base primer in a non-template directed fashion by a variety of DNA polymerases, including Taq DNA polymerase.
It has become apparent that nucleotides other than DAMP may be preferentially added to a 3xe2x80x2 end of certain PCR products in a non-template dependent manner. In particular, dGMP may often be the preferred nucleotide added. dCMP and dTMP may also be preferentially added but possibly to a lesser degree than dAMP and dGMP. Variables that may effect which nucleotide is added to the 3xe2x80x2 end of the PCR product include the primer sequences, the sequence of the target DNA and the DNA polymerase used.
The above-described cloning techniques are ineffective to directly clone PCR products that have a non-dAMP nucleotide added to one or both of the PCR product""s 3xe2x80x2 end(s).
In view of the above considerations, what is needed are compositions and methods for directly cloning PCR products which have been extended at each 3xe2x80x2 end by any nucleotide, for example, dGMP, dAMP, dCMP or dTMP.
The present invention meets this need and provides for compositions and methods that allow for the cloning of PCR products which include PCR products with any combination of the nucleotides dAMP, dTMP, dCMP or dGMP overhanging the PCR product""s 3xe2x80x2 ends.
In accordance with the present invention there are provided compositions which include one or more or two or more linear DNA molecules. These DNA molecules include a single nucleotide overhanging each 3xe2x80x2 end. The compositions may include one or more DNA molecules selected from: a linear DNA molecule with an overhanging dTMP residue at each 3xe2x80x2 end, a linear DNA molecule with an overhanging dGMP at each 3xe2x80x2 end, a linear DNA molecule with an overhanging dCMP at each 3xe2x80x2 end, a linear DNA molecule with an overhanging dAMP at each 3xe2x80x2 end, a linear DNA molecule with an overhanging dTMP at one 3xe2x80x2 end and an overhanging dAMP at another 3xe2x80x2 end, a linear DNA molecule with an overhanging dTMP at one 3xe2x80x2 end and an overhanging dGMP at another 3xe2x80x2 end, a linear DNA molecule with an overhanging dTMP at one 3xe2x80x2 end and an overhanging dCMP at another 3xe2x80x2 end, a linear DNA molecule with an overhanging dAMP at one 3xe2x80x2 end and an overhanging dGMP at another 3xe2x80x2 end, a linear DNA molecule with an overhanging dAMP at one 3xe2x80x2 end and an overhanging dCMP at another 3xe2x80x2 end and/or a linear DNA molecule with an overhanging dGMP at one 3xe2x80x2 end and an overhanging dCMP at another 3xe2x80x2 end. This embodiment allows for the cloning of PCR products with any combination of dNMPs non-specifically attached to each 3xe2x80x2 end of the PCR product.
In one embodiment, the one or more or two or more DNA molecules, which comprise a composition of the invention, are the same or substantially the same nucleotide sequence except for the single nucleotide overhanging each 3xe2x80x2 end of the DNA molecule or molecules.
In one embodiment, the one or more or two or more DNA molecules which comprise a composition of the invention are a linearized DNA molecule, for example, a linearized vector. Examples of vectors include a plasmid, a phagemid, a bacteriophage, a prokaryotic expression vector, a eukaryotic vector, a eukaryotic expression vector or a virus. In certain embodiments, the vectors may include a prokaryotic replicon, a transcription start site and/or antibiotic resistance.
Compositions of the invention may include a nucleic acid PCR product. The compositions of the invention may include being transformed into a host cell. The host cell may be eukaryotic or prokaryotic.
In one embodiment, a composition of the invention includes a ratio of DNA molecules that maximizes the percentage of positive clones in a ligation reaction between a PCR product and a composition of the invention which includes two or more linear DNA molecules.
Further in accordance with the present invention, there is provided a composition which includes a linear DNA molecule which has an overhanging dTMP at one 3xe2x80x2 end and an overhanging dCMP at another 3xe2x80x2 end. This embodiment may include a linear DNA molecule with an overhanging dCMP at each 3xe2x80x2 end and/or a linear DNA molecule with an overhanging dTMP at each 3xe2x80x2 end. This embodiment allows for the cloning of PCR products non-specifically extended at each end with any combination of DAMP and dGMP which may be the most common nucleotides added non-specifically to the 3xe2x80x2 end of PCR products.
Also included within the scope of the present invention are methods for using the compositions of the present invention to clone a DNA molecule which is produced by a polymerase chain reaction (PCR). These methods may include subjecting a DNA molecule to a polymerase chain reaction amplification to produce DNA molecules. The DNA molecules may include a DNA molecule with an overhanging dTMP residue at each 3xe2x80x2 end, a DNA molecule with an overhanging dGMP at each 3xe2x80x2 end, a DNA molecule with an overhanging dCMP at each 3xe2x80x2 end, a DNA molecule with an overhanging dAMP at each 3xe2x80x2 end, a DNA molecule with an overhanging dTMP at one 3xe2x80x2 end and an overhanging dAMP at another 3xe2x80x2 end, a DNA molecule with an overhanging dTMP at one 3xe2x80x2 end and an overhanging dGMP at another 3xe2x80x2 end, a DNA molecule with an overhanging dTMP at one 3xe2x80x2 end and an overhanging dCMP at other 3xe2x80x2 end, a DNA molecule with an overhanging dAMP at one 3xe2x80x2 end and an overhanging dGMP at another 3xe2x80x2 end, a DNA molecule with an overhanging dAMP at one 3xe2x80x2 end and an overhanging dCMP at another 3xe2x80x2 end and/or a DNA molecule with an overhanging dGMP at one 3xe2x80x2 end and an overhanging dCMP at another 3xe2x80x2 end or mixtures thereof or combinations thereof.
The method further includes the step of ligating the DNA molecules to a composition of the present invention. In one embodiment, the method further includes the step of transforming these ligated DNA molecules into a host cell.
Still further in accordance with the present invention there is provided a kit for direct cloning of polymerase chain reaction amplified nucleic acids. The kit includes a container which includes one or more or two or more linear DNA molecules which are capable of self replication in a host cell when circularized, each of the linear DNA molecules being selected from the group consisting of a linear DNA molecule with an overhanging dTMP residue at each 3xe2x80x2 end, a linear DNA molecule with an overhanging dGMP at each 3xe2x80x2 end, a linear DNA molecule with an overhanging dCMP at each 3xe2x80x2 end, a linear DNA molecule with an overhanging dAMP at each 3xe2x80x2 end, a linear DNA molecule with an overhanging dTMP at one 3xe2x80x2 end and an overhanging dAMP at another 3xe2x80x2 end, a linear DNA molecule with an overhanging dTMP at one 3xe2x80x2 end and an overhanging dGMP at another 3xe2x80x2 end, a linear DNA molecule with an overhanging dTMP at one 3xe2x80x2 end and an overhanging dCMP at another 3xe2x80x2 end, a linear DNA molecule with an overhanging DAMP at one 3xe2x80x2 end and an overhanging dGMP at another 3xe2x80x2 end, a linear DNA molecule with an overhanging DAMP at one 3xe2x80x2 end and an overhanging dCMP at another 3xe2x80x2 end and/or a linear DNA molecule with an overhanging dGMP at one 3xe2x80x2 end and an overhanging dCMP at another 3xe2x80x2 end.
Any combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art.
Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.
Base Pair (bp) is a hydrogen bonded nucleotide pair. For example, a base pair may be dAMP (A) paired with dTMP (T), or of dCMP (C) paired with dGMP (G) in a double stranded DNA molecule. In RNA, uracil (U) may be substituted for dTMP. Single stands of DNA or RNA are said to be xe2x80x9ccomplementaryxe2x80x9d when the base pairs of each DNA strand may form base pairs with the other DNA or RNA strand when the two DNA or RNA strands adopt a double stranded configuration with each other.
xe2x80x9cCleavexe2x80x9d means to enzymatically digest, for example, with a restriction endonuclease, a DNA, for example, a vector.
xe2x80x9cClonexe2x80x9d means to insert a DNA sequence, for example, a target DNA sequence into a recipient vector of the invention.
xe2x80x9cComplementaryxe2x80x9d means one or more nucleotides in a single strand of RNA or DNA having the ability to form a hydrogen bonded nucleotide pair with one or more nucleotides in another single strand of RNA or DNA.
A conserved nucleotide sequence is a nucleotide sequence that is conserved with respect to a pre-selected sequence if it non-randomly hybridizes to an exact complement of the pre-selected sequence.
xe2x80x9cCombinationsxe2x80x9d means mixtures or a sequential use of compositions. An example of a sequential use is the use of one composition followed by use of another composition. Each composition may be a specified composition.
A dNMP is a deoxynucleotide monophosphate. A dNDP is a deoxynucleotide diphosphate. A dNTP is a deoxynucleotide triphosphate. Any and all dNMPs discussed within the scope of the present invention are also contemplated as a being either a dNMP, a dNDP or a dNTP in the same or similar circumstance. Examples of dNMPs are dAMP (deoxy-adenosine monophospate) which may be referred to as A, dTMP (deoxy-thymidime monophospate) which may be referred to as T, dGMP (deoxy-guanosine monophospate) which may be referred to as G and dCMP (deoxy-cytodine monophospate) which may be referred to as C.
A duplex DNA is a double-stranded nucleic acid molecule comprising two strands of substantially complementary polynucleotides held together by one or more hydrogen bonds between each of the complementary bases present in a base pair of the duplex. Because the nucleotides that form a base pair can be either a ribonucleotide base or a deoxyribonucleotide base, the phrase xe2x80x9cduplex DNAxe2x80x9d refers to either a DNA-DNA duplex comprising two single strands of DNA (ds DNA), or an RNA-DNA duplex comprising one DNA strand and one RNA strand, or an RNA-RNA duplex comprising two single strands of RNA (ds RNA).
A gene coding sequence is a nucleic acid whose nucleotide sequence codes for an RNA or a protein molecule. Genes may comprise uninterrupted sequences of nucleotides or they may include such intervening segments as introns, promoter regions, splicing sites and repetitive sequences. A gene can be either RNA or DNA.
Hybridization is the pairing of complementary nucleotide sequences (strands of nucleic acid) to form a duplex, heteroduplex, or complex containing more than two single-stranded nucleic acids, by establishing hydrogen bonds between/among complementary base pairs. Hybridization is a specific, i.e. non-random, interaction between/among complementary polynucleotides that can be competitively inhibited.
A nucleotide is a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate group, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1xe2x80x2 carbon of the pentose) and that combination of base and sugar is a nucleoside. When the nucleoside contains a phosphate group bonded to the 3xe2x80x2 or 5xe2x80x2 position of the pentose it is referred to as a nucleotide. A sequence of operatively linked nucleotides is typically referred to herein as a xe2x80x9cbase sequencexe2x80x9d or xe2x80x9cnucleotide sequencexe2x80x9d, and their grammatical equivalents, and is represented herein by a formula whose left to right orientation is in the conventional direction of 5xe2x80x2-terminus to 3xe2x80x2-terminus. Nucleotides include nucleotide analogs.
A nucleotide analog is a purine or pyrimidine nucleotide that differs structurally from an A, T, G, C, or U base, but is sufficiently similar to substitute for the normal nucleotide in a nucleic acid molecule. Inosine (I) is a nucleotide analog that can hydrogen bond with any of the other nucleotides, A, T, G, C, or U. In addition, methylated bases are known that can participate in nucleic acid hybridization. Analogs of nucleotides are within the scope of the present invention for use in vectors comprising compositions of the present invention or in target sequences for use in accordance with the present invention.
A phagemid is a circular DNA molecule that includes one or more portions of a bacteriophage and one or more portions of a plasmid.
A polynucleotide is a polymer of single or double stranded nucleotides. As used herein xe2x80x9cpolynucleotidexe2x80x9d and its grammatical equivalents will include the full range of nucleic acids. A polynucleotide will typically refer to a nucleic acid molecule comprised of a linear strand of two or more deoxyribonucleotides and/or ribonucleotides. The exact size will depend on many factors, which in turn depends on the ultimate conditions of use, as is well known in the art. The polynucleotides of the present invention include primers, probes, RNA/DNA segments, oligonucleotides (relatively short polynucleotides), genes, vectors, plasmids, and the like.
Positive clones are cloned vectors which have a desired PCR product ligated to each end of the vector.
A primer is a polynucleotide, whether purified from a nucleic acid restriction digest or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a template nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization, such as DNA polymerase, reverse transcriptase and the like, under suitable temperature and pH reaction conditions.
Recombinant DNA is a DNA molecule produced by covalently linking a nucleic acid sequence, such as a gene, to a vector, for example, a DNA molecule sequence of the present invention. Thus, a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature rDNA""s not having the same biological origin as the host are said to be xe2x80x9cheterologousxe2x80x9d.
xe2x80x9cSubstantiallyxe2x80x9d means mostly. For example, substantially may mean more than 50%, more than 60% more than 70%, more than 80%, more than 90%, more than 99%, or more than 99.9%.
xe2x80x9cTarget DNA sequencexe2x80x9d means a duplex segment of DNA which includes one or more overhanging nucleotides. For example, a target DNA sequence may be a PCR DNA product with a single overhanging nucleotide attached to each 3xe2x80x2 end.
xe2x80x9cTemplatexe2x80x9d or xe2x80x9ctemplate sequencexe2x80x9d is a nucleic acid sequence that primers hybridize to and may also include the nucleic acid sequence amplified in a PCR reaction.
xe2x80x9cuMxe2x80x9d means micromolar, xe2x80x9culxe2x80x9d means microliter, and xe2x80x9cugxe2x80x9d means microgram.
A vector is a double stranded DNA molecule capable of autonomous replication in a cell. A heterologous DNA segment can be cloned or covalently linked to the vector so as to bring about replication of the cloned or attached DNA segment. Vectors include plasmids, phagemids, bacteriophage and viruses. Vectors capable of directing the expression of genes encoding for one or more proteins are referred to herein as xe2x80x9cexpression vectorsxe2x80x9d. Vectors may allow cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase. Vectors may include one or more replicons, one or more antibiotic resistant genes (for example, ampicillin resistance), one or more promoter sequences, the gene encoding a portion of a beta-galactosidase, for example, the LacZ portion, reporter genes, genes encoding florescent substances, genes encoding phosphorescent compounds and/or an f1 origin of replication.